The specific aim of this proposal is to solve the three-dimensional x-ray crystallographic structure of a hammerhead ribozyme. The hammerhead is one of a modest number of catalytic RNA motifs that have been characterized in recent years. Since its cleavage activity can be targeted to specific sequences, the hammerhead ribozyme is being developed as a therapeutic anti-RNA agent against viral pathogens such as HIV. Extensive mutagenesis and chemical modification studies have identified a conserved enzyme core and functional groups required for hammerhead activity, but have not yet elucidated a mechanism of catalysis; knowledge of the three-dimensional structure will be necessary for understanding the enzymatic mechanism. Structural studies will be pursued on two fronts simultaneously: First: Crystals of two hammerhead RNA-DNA complexes have been grown; the most promising crystal form is trigonal, space group P31-21, a = 92.5 Angstroms, c = 185.0 Angstroms, and diffracts to approximately 3.2 Angstroms resolution. The structure will be solved by multiple isomorphous replacement; this will reveal the overall tertiary fold of the hammerhead. Second: A modest number (3-5) of other hammerhead constructs which may show significant structural differences from the RNA-DNA complex will be synthesized and crystallized. These will include (i) a hammerhead complex which is all RNA except for a single deoxyribonucleotide at the cleavage site to inhibit cleavage; (ii) a hammerhead "minizyme" in which one of the duplex stems that appears to be nonessential for activity is deleted; and (iii) a mostly-deoxy hammerhead in which only the minimum number of ribonucleotides required for activity are incorporated into an otherwise all-DNA enzyme.